Using state-of-the-art genome editing tools, such as Cas9 protein and synthetic guide RNAs, the genome of human induced pluripotent stem cells (hiPSC) can now be easily edited to introduce genetic defects related to disease. These genome edited hiPSC can then be differentiated into for example cardiomyocytes or neurons, which can be implemented to model disease in vitro for basic research or drug discovery. While in general genome editing of hiPSC has become standard practice, higher throughput, larger scale and consistent generation of genome edited hiPSC lines remains challenging due to the complex nature of hiPSC culture conditions. Furthermore, manual picking of hundreds of colonies to identify clonal lines carrying the desired genomic edit remains a labor intensive process. We therefore sought to implement automation to standardize and facilitate key steps covering the majority of the genome editing workflow in hiPSCs.

Of the required steps to generate a clonal, genome edited hiPSC line, we so far achieved full automation of single cell seeding, expansion and consolidation, simply relying on available hiPSC products, a cell sorter and a simple liquid handler. Using SNP introductions to model disease as a genome editing example in hiPSC, we demonstrate, using these automated workflows, that clonal genome edited hiPSC lines can be derived reproducibly across multiple hiPSC backgrounds, with high first-time-right rates that drive throughput and scale. These methods did not affect the karyotype of the generated hiPSC lines, which furthermore maintained their typical pluripotency characteristics and potential to differentiate into specialized cells, allowing us to study the biology of the genomic changes made.

By implementing existing and novel innovations in the hiPSC product space, such as StemFlexTM hiPSC medium, RevitaCellTM supplement and rhLaminin-521 matrix, we identified automated ways to drive the throughput, scale and consistency of the genome editing workflow in hiPSC.